Production of penicillin mold and jelly



. the application ;filed February Patented Sept. 2, 1952 No Drawing."Application September 6,1951,

- ;-SerialNo.24'5,41'5

'- 10' carat. (oi. 167 58) 'iGranted under the act iof..March 3, 1883,as amendedA-priIBO, 1928;. 370. 0.16. "757) ".i'Il'relnventionhereindescribed'may be manu- Iactured and :used by. or for theGovernmentof theUnited States f America for governmental purposesthroughout the worldwithout. the paymerit tome of any royalty thereon.

. This application is a continuation-in-part-of 10, 1950 and bearingSerialNo. 143,610.

:This invention relates to the production of an activated penicillinproducing' mold and to a process of culturing'this mold to produce anaqueous mixture containing idissolved penicillin. Itafu'rther-relates.to the production of a penicillin jelly :for use in topical" therapy;particularly a jelly produced byxthe emulsification of the aforesaidaqueous :mixture containing penicillin and a solidifying agent. ;;In'general; the. invention involves-culturing P. notatwm' :in a sterileculture medium containing oxidized potato .juice to produce an activatedmold. Thereafterythe activated mold is cultured'in a conventional mannerto produce an aqueous :mixture ,containing dissolved penicillin and themixture :is :emulsified'with a solidifyin agent to form a jelly.

' .Accordingtothe. invention, .1. notatumpreferably a certified strainthereof although any strain iS'OPBlflfil/QlS cultured .in a culturemedium containing oxidized-potato juice to produce an activated mold.Except for the oxidized-potato juice,

such as agarg'is also included tosolidify themedium when it is to ,beused. as .a surface culture medium. The medium is sterilized, prior toits use, in accordance with conventional practice.

.The, production of the activatedmold will now be described-- ingreater. detail, utilizing a solid, sterile culturemedium, that is, asurface culture medium as indicated above, to cuture P. notatum.

This solid; sterile culture medium is prepared Preparation of oxidizedpotato iuice.--Raw po tatoes are thoroughly scrubbed, pared, weighed,and ground. To each part, by weight, of the ground potato is added twoparts, by weight, of

distilled water, and the resulting mixture is exthroughout. The stirredmixture lfis then filtered through cheesecloth to :permit the oxidized:potato'juice-and the starch to pass through, and the filtrate'iscollected inflasks. The' flasks-are autoclaved for one-hour; at"l15Cyand allowed to stand over-night; whereupon the cooked starchprecipitates. The'oxidized juice is'ifseparated from the precipitatedstarch by decan'tation and filtered through cotton. Nowater should 'beadded to thexresulting filtrate-of oxidized juice to mal ie uptovolume.1

P To the oxidized juice 'is added 2% of dextrose as the sugar, and 4% ofagar as the solidifying agent. The resulting mixture is cooked forfo'nehour and filteredth'reugh cotton. Thej'filtrate is collected in smallflasks-and autoclaved 'for 20 minutes-atllfl jc. Uponcooling, thesolidjsterile 10 to 14 days, to produce the activated mold which isreadily removed from'the surfaceof the medium."

The activatedmold is then cultured one conventional culture'medium asfor example unoxidiz-edpotato juice, to produce penicillin in'aque-Que-mixture which "may subsequently be emulsified-into a jelly by addinga solidifying-agent} particularly agar, and mechanically emulsifying thethixture. These procedures are exhibited in greater-detailas follows: 9

At the end of the first culture periodfthe re sulting activated mold istransferredto the sec ond' sterileiculture-mediurn containing"the unoxidized potato juice, and is permittedPto grow on this-medium to"produce penicillin, preferably for a period of'a-boutirom 1-0 tolfdaysi- A'sa result, the peni'cillim'which is produced 'as-the moldexudate, is dissolvedint-he supernatant water er the -medium, this waterbeing" covered with'mold growth.

'This second medium is preparedas follovirsi Rawpotatoes are thoroughlyscrubbed, pared;

snej eut into cy'l-inders--or small pieces. The cut potato isimmediately placed in an -Erlenmeyer flask, and water is added theretoin the proportion of 3 parts, by weight, of water to each part, byweight, of potato. The flask is plugged with cotton and autoclaved for20 minutes at C. The effect of this treatment is to substantiallyprevent oxidation of the potato. v

At the end of this second culture period, the resulting yellow liquid(penicillin in solution) along with the covering mold is poured on athird solid, sterile culture medium containing unoxlcovered withdistilled water to prevent oxidation of the potato, the resultingmixture containing" water and potato in the proportion olf'21tiarts, byWeight, of water to each part, by weight, :or The mixture is steamed forminutes potato. or permitted to simmer in a water bath at a temperaturebelow 60 C. for'one -hour,1-after;.1 which it is filtered throughcheesecloth andthe I unoxidized potato juice filtrate made up the,.

original amount with water.

To the unoxidized potato juice filtratethe'rev is added 2% of dextroseas the sugar, and '4% agar as the solidifying agent, and the resultingmixture cooked for one hour and filtered through cotton. The resultingfiltrate is then autoclaved for 20 minutes at 115 C. and upon cooling,the solid, sterile culture mediumis obtained.

After the third culture period, the arising mold may be removed, and theremaining part of the culture, which contains an aqueous solution ofpenicillin in agar, is mechanically-emulsified, as by being forcedthrough a sieve, to a jelly.

Th product has good keeping qualities, probably due to the agar base,and is well-maintained at refrigeration temperature.- The penicillinunitage of the product is from to 50 Oxford units per gram. I v Theproduct of the invention appears to be a slowreleasevehicle, anadvantage similar to the concentration of penicillin in beeswax andpeanut oil. Attestation to its stability on hospital shelves for periodsexceedingB months has been made. Chemical tests have shown that-it isnontoxic.

Having thus described my invention, I claim:

1. A process comprising culturing P. notatum on a sterile culture mediumcontaining oxidized potato juice to produce an activated mold. 2. Aprocess comprising culturing P. notatum ona solid, sterile culturemedium containing oxidized potato juice and dextrose to produce anactivated mold.

-3 A process comprising culturing P. notatum on a solid, sterile culturemedium containing oxidizedpotato juice, dextrose,=and agar to produce anactivated mold.

4. A-process comprising culturing P. notatum on a sterile culture mediumcontaining oxidized potato juice to produce an activated mold andculturing the activated mold on a second sterile culture medium toproduce penicillin.

5. A process comprising culturing P. notatum ona solid, sterile culturemedium containing oxidized potato juice and dextrose to produce anactivated mold and culturing the activated mold on, a second sterileculture medium containing unoxidized potato juice to produce an aqueousmixture containing mold and dissolved penicillin.

6. A process comprising culturing P. notatum on a solid, sterile culturemedium containing oxidized potato juice and dextrose to produce anactivated mold and culturing the activated mold on a solid, sterileculture medium containing unoxidized potato: juice and dextrose andallowing the mold to grow there in'to produce penicillin.

7. A process comprising culturing P. notatum 'on a. solid, sterileculture medium containing 10" activated mold, culturing the activatedmold on oxidized potato juice and dextrose to produce an a secondsterile culture medium containing unoxidized potato juice to produce anaqueous mixu-ture containing .mold and dissolved penicillin,

transferring the aqueous mixture to a third solid, sterile culturemedium containing unoxidized potato juice and dextrose and allowing themold to grow thereon to produce penicillin.

' 8. A process comprising culturing P. notatum on a solid, sterilecurture medium containing oxidized potato juice and dextrose to producean activated mold, culturing the activated mold on a second sterileculture medium containing un= oxidized potato juice to produce anaqueous mixture containing mold and dissolved penicillin, transferringthe aqueous mixture to a thirds'olid, sterile culture mediumcontaini'ngunoxidized potato juice and dextrose and allowing the mold togrow thereon to produce pencillin, removing the arising mold, andmechanically emulsifying the remainder of the culture. medium .to ajelly.

9. A process comprising culturing P. notatum on a solid, sterile culturemedium containing oxidized potato juice, dextrose, and agar-to producean activated mold, culturin'gthe activated mold on a second sterileculture medium containing unoxidized potato juice to produ'cean aqueousmixture containing mold and dissolved penicillin, transferring theaqueous mixture to a third solid, sterile culture medium containingunoxidized potato juice, dextrose, and agar and allowing the mold togrow thereon to produce penicillin, removing the arising mold,andmechanically emulsifying the remainder of the cul' ture medium to ajelly.

10. A process comprising culturing a penicillin-producing mold on asterile culture medium to produce penicillin, said mold having beenproduced by culturing P. notatum on a sterile culture medium containingoxidized potato juice.

' AGNES J. QUIRK.

REFERENCES CITED The following references are of record in the file ofthis patent: v UNITED STATES PATENTS OTHER REF R NC S". i

. Hazeltine: The Washington Post, October 3,

1951, page 14. article relating to 1Athletes Foot

9. A PROCESS COMPRISING CULTURING P. NOTATUM ON A SOLID, STERILE CULTUREMEDIUM CONTAINING OXIDIZED POTATO JUICE, DEXTROSE, AND AGAR TO PRODUCEAN ACTIVATED MOLD, CULTURING THE ACTIVATED MOLD ON A SECOND STERILECULTURE MEDIUM CONTAINING UNOXIDIZED POTATO JUICE TO PRODUCE AN AQUEOUSMIXTURE CONTAINING MOLD AND DISSOLVED PENICILLIN, TRANSFERRING THEAQUEOUS MIXTURE TO A THIRD SOLID, STERILE CULTURE MEDIUM CONTAININGUNOXIDIZED POTATO JUICE, DEXTRODE, AND AGAR AND ALLOWING THE MOLD TOGROW THEREON TO PRODUCE PENICILLIN, REMOVING THE ARISING MOLD, ANDMECHANICALLY EMULSIFYING THE REMAINDER OF THE CULTURE MEDIUM TO A JELLY.